Difference between revisions of "Part:BBa K3075000"
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==== Introduction ====  
 
==== Introduction ====  
  
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PAM-SnoopT consists of the enzyme Phenylalanine aminomutase (PAM) fused at the C-terminus to a short polypeptide tag (Snooptag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of PAM originating from Taxus wallichiana var. chinensis was utilised. (1)
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PAM-Snooptag-His consists of the enzyme Phenylalanine aminomutase (PAM) fused at the C-terminus to a short polypeptide tag (Snooptag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of PAM originating from ''Taxus wallichiana var. chinensis'' was utilised. (1) The SnoopTag is a small polypeptide tag that spontaneously forms an isopeptide bond between reactive amino acid side chains to its corresponding SnoopCatcher (Brune, 2017). This system opens up a variety of applications, utilising the catcher-tag conjugation system for bioconjugation and synthetic assembly of the DBAT enzyme to SnoopCatcher containing proteins.
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Image
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The Hexahistidine tag is a common additive due to its high affinity for metal ions used in the purification technique of immobilized metal affinity chromatography (IMAC). Ni2+ ions were used for his-tag purification due to its high yield.
  
 
==== Usage and Biology ====
 
==== Usage and Biology ====
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Phenylalanine aminomutase catalyses the conversion of 2S-α-phenylalanine to 3R-β-phenylalanine. (2) Naturally, this enzyme is involved in the synthesis of trans-cinnamate from L-phenylalanine, an important part of Phenylpropanoid metabolism. It is also known to catalyse the initial step of N-benzoyl phenylisoserinoyl biosynthesis, which functions as the side chain of the anticancer drug Paclitaxel. (1) Recombinant phenylalanine aminomutase has a sequence of 698 amino acid residues with a molecular mass of 76,530 Daltons and maintains stability at a pH of 9-11 and temperature of 60-70C.  
 
Phenylalanine aminomutase catalyses the conversion of 2S-α-phenylalanine to 3R-β-phenylalanine. (2) Naturally, this enzyme is involved in the synthesis of trans-cinnamate from L-phenylalanine, an important part of Phenylpropanoid metabolism. It is also known to catalyse the initial step of N-benzoyl phenylisoserinoyl biosynthesis, which functions as the side chain of the anticancer drug Paclitaxel. (1) Recombinant phenylalanine aminomutase has a sequence of 698 amino acid residues with a molecular mass of 76,530 Daltons and maintains stability at a pH of 9-11 and temperature of 60-70C.  
  
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==== Characterisation ====
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Image
  
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The PAM-SnoopT gBlock was synthesised by IDT. PAM-SnoopT was ligated into pET19b plasmid backbone by Gibson assembly and transformed into competent T7 express E.coli cells. Colony PCR was performed using primers listed below and the amplicon was visualised by gel electrophoresis.
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==== Characterisation ====
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Primers used:
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- T7 Forward : 5’-TAATACGACTCACTATAGGG
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- T7 Reverse : 5’-GCTAGTTATTGCTCAGCGG
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'''Protein expression assay'''
  
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A small scale grow up of colonies was performed and plasmid DNA was extracted via a QIAGEN miniprep kit. Miniprepped samples were visualised via gel electrophoresis (Figure 1) and submitted for sequencing by the Ramaciotti Centre for Genomics (Figure 2).  
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Cells containing a plasmid with the PAM insert (as confirmed by colony PCR and sequencing) were grown up and a sample of this was used to perform a protein expression assay. Bug buster was used to separate soluble and insoluble proteins.
  
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Starter culture was made for colonies of interest and large-scale grow up was done. Expression of the recombinant protein was induced using IPTG and harvested cells were lysed via Sonication. The His-tagged protein was then purified via IMAC. Results are shown on figure 3.
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Figure ?. Protein expression assay using bug buster to determine expression of Phenylalanine aminomutase (PAM) as soluble and insoluble form.

Revision as of 04:26, 19 October 2019

PAM-SnoopT

Introduction

PAM-Snooptag-His consists of the enzyme Phenylalanine aminomutase (PAM) fused at the C-terminus to a short polypeptide tag (Snooptag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of PAM originating from Taxus wallichiana var. chinensis was utilised. (1) The SnoopTag is a small polypeptide tag that spontaneously forms an isopeptide bond between reactive amino acid side chains to its corresponding SnoopCatcher (Brune, 2017). This system opens up a variety of applications, utilising the catcher-tag conjugation system for bioconjugation and synthetic assembly of the DBAT enzyme to SnoopCatcher containing proteins.

Image

The Hexahistidine tag is a common additive due to its high affinity for metal ions used in the purification technique of immobilized metal affinity chromatography (IMAC). Ni2+ ions were used for his-tag purification due to its high yield.

Usage and Biology

Phenylalanine aminomutase catalyses the conversion of 2S-α-phenylalanine to 3R-β-phenylalanine. (2) Naturally, this enzyme is involved in the synthesis of trans-cinnamate from L-phenylalanine, an important part of Phenylpropanoid metabolism. It is also known to catalyse the initial step of N-benzoyl phenylisoserinoyl biosynthesis, which functions as the side chain of the anticancer drug Paclitaxel. (1) Recombinant phenylalanine aminomutase has a sequence of 698 amino acid residues with a molecular mass of 76,530 Daltons and maintains stability at a pH of 9-11 and temperature of 60-70C.

Image

Characterisation

Protein expression assay

Cells containing a plasmid with the PAM insert (as confirmed by colony PCR and sequencing) were grown up and a sample of this was used to perform a protein expression assay. Bug buster was used to separate soluble and insoluble proteins.

Figure ?. Protein expression assay using bug buster to determine expression of Phenylalanine aminomutase (PAM) as soluble and insoluble form.